Fetal oxidative stress mechanisms of neurodevelopmental deficits and exacerbation by ethanol and methamphetamine

Shapiro A.M., Miller-Pinsler L., Wells P.G.

The breast cancer 1 (brca1) gene is associated with breast and ovarian cancers, and heterozygous (+/-) brca1 knockout progeny develop normally, suggesting a negligible developmental impact. However, our results show BRCA1 plays a broader biological role in protecting the embryo from oxidative stress. Sox2-promoted Cre-expressing hemizygous males were mated with floxed brca1 females, and gestational day 8 +/- brca1 conditional knockout embryos with a 28% reduction in protein expression were exposed in culture to the reactive oxygen species (ROS)-initiating drug ethanol (EtOH). Untreated +/- brca1-deficient embryos developed normally, but when exposed to EtOH exhibited increased levels of oxidatively damaged DNA, measured as 8-oxo-2'-deoxyguanosine, γH2AX, which is a marker of DNA double strand breaks that can result from 8-oxo-2'-deoxyguanosine, formation, and embryopathies at EtOH concentrations that did not affect their brca1-normal littermates. These results reveal that even modest BRCA1 deficiencies render the embryo more susceptible to drug-enhanced ROS formation, and corroborate a role for DNA oxidation in the mechanism of EtOH teratogenesis.

Dang D., Shin E., Nam Y., Ryoo S., Jeong J.H., Jang C., Nabeshima T., Hong J., Kim H.

Activation of NADPH oxidase (PHOX) plays a critical role in mediating dopaminergic neuroinflammation. In the present study, we investigated the role of PHOX in methamphetamine (MA)-induced neurotoxic and inflammatory changes in mice. We examined changes in mitogen-activated protein kinases (MAPKs), mitochondrial function [i.e., mitochondrial membrane potential, intramitochondrial Ca2+ accumulation, mitochondrial oxidative burdens, mitochondrial superoxide dismutase expression, and mitochondrial translocation of the cleaved form of protein kinase C delta type (cleaved PKCδ)], microglial activity, and pro-apoptotic changes [i.e., cytosolic cytochrome c release, cleaved caspase 3, and terminal deoxynucleotidyl transferase dUDP nick-end labeling (TUNEL) positive populations] after a neurotoxic dose of MA in the striatum of mice to achieve a better understanding of the effects of apocynin, a non-specific PHOX inhibitor, or genetic inhibition of p47phox (by using p47phox knockout mice or p47phox antisense oligonucleotide) against MA-induced dopaminergic neurotoxicity. Phosphorylation of extracellular signal-regulated kinases (ERK1/2) was most pronounced out of MAPKs after MA. We observed MA-induced phosphorylation and membrane translocation of p47phox in the striatum of mice. The activation of p47phox promoted mitochondrial stresses followed by microglial activation into the M1 phenotype, and pro-apoptotic changes, and led to dopaminergic impairments. ERK activated these signaling pathways. Apocynin or genetic inhibition of p47phox significantly protected these signaling processes induced by MA. ERK inhibitor U0126 did not exhibit any additional positive effects against protective activity mediated by apocynin or p47phox genetic inhibition, suggesting that ERK regulates p47phox activation, and ERK constitutes the crucial target for apocynin-mediated inhibition of PHOX activation. Our results indicate that the neuroprotective mechanism of apocynin against MA insult is via preventing mitochondrial burdens, microglial activation, and pro-apoptotic signaling process by the ERK-dependent activation of p47phox.

Bjørge M., Hildrestrand G., Scheffler K., Suganthan R., Rolseth V., Kuśnierczyk A., Rowe A., Vågbø C., Vetlesen S., Eide L., Slupphaug G., Nakabeppu Y., Bredy T., Klungland A., Bjørås M.

Varadinova M., Boyadjieva N.

The etiology of autism spectrum disorders (ASDs) still remains unclear and seems to involve a considerable overlap between polygenic, epigenetic and environmental factors. We have summarized the current understanding of the interplay between gene expression dysregulation via epigenetic modifications and the potential epigenetic impact of environmental factors in neurodevelopmental deficits. Furthermore, we discuss the scientific controversies of the relationship between prenatal exposure to alcohol and alcohol-induced epigenetic dysregulations, and gene expression alterations which are associated with disrupted neural plasticity and causal pathways for ASDs. The review of the literature suggests that a better understanding of developmental epigenetics should contribute to furthering our comprehension of the etiology and pathogenesis of ASDs and fetal alcohol spectrum disorders.

Ali K., Mahjabeen I., Sabir M., Mehmood H., Kayani M.A.

In first part of this study association between OGG1 polymorphisms and breast cancer susceptibility was explored by meta-analysis. Second part of the study involved 925 subjects, used for mutational analysis of OGG1 gene using PCR-SSCP and sequencing. Fifteen mutations were observed, which included five intronic mutations, four splice site mutations, two 3′UTR mutations, three missense mutations, and a nonsense mutation. Significantly (p<0.001) increased (~29 fold) breast cancer risk was associated with a splice site variant g.9800972T>G and 3′UTR variant g.9798848G>A. Among intronic mutations, highest (~15 fold) increase in breast cancer risk was associated with g.9793680G>A (p<0.009). Similarly ~14-fold increased risk was associated with Val159Gly (p<0.01), ~17-fold with Gly221Arg (p<0.005), and ~18-fold with Ser326Cys (p<0.004) in breast cancer patients compared with controls, whereas analysis of nonsense mutation showed that ~13-fold (p<0.01) increased breast cancer risk was associated with Trp375STOP in patients compared to controls. In conclusion, a significant association was observed between OGG1 germ line mutations and breast cancer risk. These findings provide evidence that OGG1 may prove to be a good candidate of better diagnosis, treatment, and prevention of breast cancer.

Miller-Pinsler L., Wells P.G.

Reactive oxygen species (ROS) have been implicated in the mechanism of ethanol (EtOH) teratogenicity, but the protective role of the embryonic antioxidative enzyme catalase is unclear, as embryonic activity is only about 5% of maternal levels. We addressed this question in a whole embryo culture model. C57BL/6 mouse embryos expressing human catalase (hCat) or their wild-type (C57BL/6 WT) controls, and C3Ga.Cg-Cat(b)/J catalase-deficient, acatalasemic (aCat) mouse embryos or their wild-type C3HeB/FeJ (C3H WT) controls, were explanted on gestational day (GD) 9 (plug=GD 1), exposed for 24h to 2 or 4mg/mL EtOH or vehicle, and evaluated for functional and morphological changes. hCat and C57BL/6 WT vehicle-exposed embryos developed normally, while EtOH was embryopathic in C57BL/6 WT embryos, evidenced by decreases in anterior neuropore closure, somites developed, turning and head length, whereas hCat embryos were protected (p

Xiang Y., Kim K., Gelernter J., Park I., Zhang H.

Chronic alcohol consumption may result in sustained gene expression alterations in the brain, leading to alcohol abuse or dependence. Because of ethical concerns of using live human brain cells in research, this hypothesis cannot be tested directly in live human brains. In the present study, we used human embryonic stem cell (hESC)-derived cortical neurons as in vitro cellular models to investigate alcohol-induced expression changes of genes involved in alcohol metabolism (ALDH2), anti-apoptosis (BCL2 and CCND2), neurotransmission (NMDA receptor subunit genes: GRIN1, GRIN2A, GRIN2B, and GRIN2D), calcium channel activity (ITPR2), or transcriptional repression (JARID2). hESCs were differentiated into cortical neurons, which were characterized by immunostaining using antibodies against cortical neuron-specific biomarkers. Ethanol-induced gene expression changes were determined by reverse-transcription quantitative polymerase chain reaction (RT-qPCR). After a 7-day ethanol (50 mM) exposure followed by a 24-hour ethanol withdrawal treatment, five of the above nine genes (including all four NMDA receptor subunit genes) were highly upregulated (GRIN1: 1.93-fold, P = 0.003; GRIN2A: 1.40-fold, P = 0.003; GRIN2B: 1.75-fold, P = 0.002; GRIN2D: 1.86-fold, P = 0.048; BCL2: 1.34-fold, P = 0.031), and the results of GRIN1, GRIN2A, and GRIN2B survived multiple comparison correction. Our findings suggest that alcohol responsive genes, particularly NMDA receptor genes, play an important role in regulating neuronal function and mediating chronic alcohol consumption-induced neuroadaptations.

Vaclová T., Gómez-López G., Setién F., Bueno J.M., Macías J.A., Barroso A., Urioste M., Esteller M., Benítez J., Osorio A.

BRCA1 germline mutations increase the lifetime risk of developing breast and ovarian cancers. However, taking into account the differences in disease manifestation among mutation carriers, it is probable that different BRCA1 mutations have distinct haploinsufficiency effects and lead to the formation of different phenotypes. Using lymphoblastoid cell lines derived from heterozygous BRCA1 mutation carriers and non-carriers, we investigated the haploinsufficiency effects of various mutation types using qPCR, immunofluorescence, and microarray technology. Lymphoblastoid cell lines carrying a truncating mutation showed significantly lower BRCA1 mRNA and protein levels and higher levels of gamma-H2AX than control cells or those harboring a missense mutation, indicating greater spontaneous DNA damage. Cells carrying either BRCA1 mutation type showed impaired RAD51 foci formation, suggesting defective repair in mutated cells. Moreover, compared to controls, cell lines carrying missense mutations displayed a more distinct expression profile than cells with truncating mutations, which is consistent with different mutations giving rise to distinct phenotypes. Alterations in the immune response pathway in cells harboring missense mutations point to possible mechanisms of breast cancer initiation in carriers of these mutations. Our findings offer insight into how various heterozygous mutations in BRCA1 could lead to impairment of BRCA1 function and provide strong evidence of haploinsufficiency in BRCA1 mutation carriers.

Vargesson N.

Nearly 60 years ago thalidomide was prescribed to treat morning sickness in pregnant women. What followed was the biggest man-made medical disaster ever, where over 10,000 children were born with a range of severe and debilitating malformations. Despite this, the drug is now used successfully to treat a range of adult conditions, including multiple myeloma and complications of leprosy. Tragically, a new generation of thalidomide damaged children has been identified in Brazil. Yet, how thalidomide caused its devastating effects in the forming embryo remains unclear. However, studies in the past few years have greatly enhanced our understanding of the molecular mechanisms the drug. This review will look at the history of the drug, and the range and type of damage the drug caused, and outline the mechanisms of action the drug uses including recent molecular advances and new findings. Some of the remaining challenges facing thalidomide biologists are also discussed.

Miller-Pinsler L., Sharma A., Wells P.G.

Methanol (MeOH) teratogenicity in rodents may be mediated in part by reactive oxygen species (ROS), the source of which is unknown. To determine if MeOH enhances embryonic ROS-producing NADPH oxidases (NOXs), p22phox mRNA and protein and oxidatively damaged protein were measured in gestational day 12 MeOH-exposed CD-1 mouse embryos with or without pretreatment with the free radical spin trap phenylbutylnitrone (PBN) or the NOX inhibitor diphenyleneiodonium chloride (DPI). MeOH exposure upregulated p22phox mRNA and protein expression, and enhanced protein oxidation, within 3–6 h. Compared to embryos exposed to MeOH alone, PBN and DPI pretreatment decreased MeOH-enhanced p22phox mRNA expression, DPI but not PBN blocked p22phox protein expression, and both blocked protein oxidation. To assess developmental relevance, mouse embryos were exposed in culture for 24 h to MeOH or vehicle with or without pretreatment with PBN, DPI, or the prostaglandin H synthase (PHS) inhibitor eicosatetraynoic acid (ETYA), and evaluated for abnormalities. ETYA did not prevent MeOH embryopathies, despite blocking phenytoin embryopathies (ROS-initiating positive control), precluding bioactivation of MeOH or its metabolites by PHS. Concentration-dependent MeOH embryopathies were blocked by both DPI and PBN pretreatment, suggesting that enhanced embryonic NOX-catalyzed ROS formation and oxidative stress may contribute to the mechanism of MeOH embryopathies.

Zhou F.C.

The recent study by Stepien, Lussier, Pavlidis, Kobor, and Weinberg demonstrates how prenatal alcohol exposure alters genomic expression far into the adulthood, and also provides a new view about how transcriptions might respond differently upon new environmental challenge. This study also provides a more comprehensive view by filling a gap of the global transcriptome analyses of FASD.

Zhou P., Li B., Ji J., Wang M., Gao C.

The oxyguanine glycosylase 1 (OGG1) gene has an important role in DNA repair, and the polymorphism of the gene may alter cancer susceptibility. This study aims to examine the association between the OGG1 Ser326Cys polymorphism and cancer risk based on meta-analysis. Relevant studies were identified through a search of PubMed and Weipu databases, and a total of 109 studies including 111 comparisons containing 34,041 cases and 42,730 controls were enrolled. Overall, significant association was observed between OGG1 Ser326Cys polymorphism and cancer risk in all genetic models except for heterozygote model (Cys/Cys + Cys/Ser vs Ser/Ser: OR 1.071, 95 % CI 1.019–1.125; Cys/Cys vs Cys/Ser + Ser/Ser: OR 1.159, 95 % CI 1.076–1.248; Cys/Cys vs Ser/Ser: OR 1.202, 95 % CI 1.105–1.308). In stratified analysis by cancer type, significantly increased cancer risk was observed in digestive system cancer, head and neck cancer and lung cancer. For gynecologic cancer, significantly increased cancer risk was also observed in homozygote model (OR 1.974, 95 % CI 1.254–3.107). In addition, in stratified analysis by ethnicities, increased cancer risk was found in Asians (Cys/Cys vs Cys/Ser + Ser/Ser: OR 1.195, 95 % CI 1.088–1.313; Cys/Cys + Cys/Ser vs Ser/Ser: OR 1.115, 95 % CI 1.045–1.190; Cys/Cys vs Ser/Ser: OR 1.273, 95 % CI 1.149–1.410). The OGG1 Ser326Cys polymorphism may be a risk factor for cancers of lung, digestive system and head and neck.

Miller-Pinsler L., Pinto D.J., Wells P.G.

Studies in mice with deficient antioxidative enzymes have shown that physiological levels of reactive oxygen species (ROS) can adversely affect the developing embryo and fetus. Herein, DNA repair-deficient progeny of oxoguanine glycosylase 1 ( ogg1 )-knockout mice lacking repair of the oxidative DNA lesion 8-oxo-2′-deoxyguanosine (8-oxodGuo) exhibited enhanced postnatal neurodevelopmental deficits, revealing the pathogenic potential of 8-oxodGuo initiated by physiological ROS production in fetal brain and providing the first evidence of a pathological phenotype for ogg1 -knockout mice. Moreover, when exposed in utero to ethanol (EtOH), ogg1- knockout progeny exhibited higher levels of 8-oxodGuo in fetal brain and more severe postnatal neurodevelopmental deficits than wild-type littermates, both of which were blocked by pretreatment with the free radical trapping agent phenylbutylnitrone. These results suggest that ROS-initiated DNA oxidation, as distinct from altered signal transduction, contributes to neurodevelopmental deficits caused by in utero EtOH exposure, and fetal DNA repair is a determinant of risk. • DNA repair-deficient ogg1- knockout mice have impaired neurodevelopment. • In utero ethanol (EtOH) enhances fetal DNA oxidation and impairs neurodevelopment. • Both EtOH outcomes are greater in ogg1- knockout progeny. • Both EtOH outcomes in ogg1 -knockout progeny are blocked by a free radical spin trap. • Fetal OGG1 and DNA oxidation are determinants of risk for in utero EtOH outcomes.

Shpyleva S., Ivanovsky S., de Conti A., Melnyk S., Tryndyak V., Beland F.A., James S.J., Pogribny I.P.

The molecular pathogenesis of autism is complex and involves numerous genomic, epigenomic, proteomic, metabolic, and physiological alterations. Elucidating and understanding the molecular processes underlying the pathogenesis of autism is critical for effective clinical management and prevention of this disorder. The goal of this study is to investigate key molecular alterations postulated to play a role in autism and their role in the pathophysiology of autism. In this study we demonstrate that DNA isolated from the cerebellum of BTBR T+tf/J mice, a relevant mouse model of autism, and from human post-mortem cerebellum of individuals with autism, are both characterized by an increased levels of 8-oxo-7-hydrodeoxyguanosine (8-oxodG), 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC). The increase in 8-oxodG and 5mC content was associated with a markedly reduced expression of the 8-oxoguanine DNA-glycosylase 1 (Ogg1) and increased expression of de novo DNA methyltransferases 3a and 3b (Dnmt3a and Dnmt3b). Interestingly, a rise in the level of 5hmC occurred without changes in the expression of ten-eleven translocation expression 1 (Tet1) and Tet2 genes, but significantly correlated with the presence of 8-oxodG in DNA. This finding and similar elevation in 8-oxodG in cerebellum of individuals with autism and in the BTBR T+tf/J mouse model warrant future large-scale studies to specifically address the role of OGG1 alterations in pathogenesis of autism.

Miller-Pinsler L., Wells P.G.

Reactive oxygen species (ROS) have been implicated in the teratogenicity of alcohol (ethanol, EtOH). To determine the involvement of embryonic oxidative DNA damage, DNA repair-deficient oxoguanine glycosylase 1 (ogg1) knockout embryos were exposed in culture to EtOH (2 or 4 mg/ml), with or without pretreatment with the free radical spin trap phenylbutylnitrone (PBN) (0.125 mM). Visceral yolk sacs were used to genotype embryos for DNA repair status and gender. EtOH caused a concentration-dependent decrease in anterior neuropore closure (ANPC), somite development, turning, crown–rump length (CRL), yolk sac diameter (YSD) and head length (HL) (p < 0.001) in all 3 ogg1 genotypes. There was a further ogg1 gene dose-dependent decrease from +/+ to −/− embryos in ANPC, somite development, turning, CRL and HL (p < 0.05), and a gene-dependent correlation between HL and ANPC (p < 0.01). Female embryos exhibited lesser ANPC and turning than males (p < 0.05), suggesting underlying gender-dependent target-specific determinants. PBN pretreatment increased ANPC, somite development, turning, CRL, YSD and HL (p < 0.001), although this protection against EtOH was slightly less effective in −/− embryos. Oxidatively damaged DNA determined as 8-oxo-2′-deoxyguanosine (8-oxodGuo), which is repaired by OGG1, was measured in single embryos in vivo after maternal EtOH treatment (4 g/kg i.p). EtOH increased embryonic 8-oxodGuo in an ogg1 gene-dependent fashion, with the highest levels in −/− embryos. These results show that embryonic DNA repair status and gender are determinants of risk. ROS-initiated embryonic DNA oxidation is involved in EtOH embryopathies.

Wing C.E., Foster E.G., Summerlin M., Bade A.N.

Neurodevelopment is a continuous, complex, and strictly regulated biological process that begins early after conception during embryonic life and continues into young adulthood. However, fetal brain development during gestation is a window of high vulnerability. Diverse coordinated biological processes occur during this critical period including neurulation, neurogenesis, microglial entry, synaptogenesis and pruning, programmed cell death, and myelination. Disturbances to these processes can produce short- or long-term detrimental effects on brain development. These include chronic forms of congenital malformations or long-term postnatal neurodevelopmental disorders. Congenital malformations include neural tube defects (NTDs) in which the development of the brain or the spinal cord structure is impaired. These malformations can be fatal. Moreover, impairment in biological processes can lead to a wide range of long-term functional disorders called neurodevelopmental disorders. These are a group of disorders characterized by impairments in developmental domains including cognition, communication, behavior, or motor skills. Genetic factors or environmental exposures during gestation have been linked with neurodevelopmental deficits. In this chapter, cellular and molecular pathways of neurogenesis are described in addition to characterizations of NTDs and postnatal neurodevelopmental disorders. Finally, an update on neurodevelopmental outcomes of prenatal exposure to drugs of abuse, prescribed medications, or environmental factors is provided.

Dylag K.A., Wieczorek-Stawinska W., Burkot K., Drzewiecki L., Przybyszewska K., Tokarz A., Dumnicka P.

Background/Objectives: Malnutrition is a significant concern in paediatric populations, particularly among children with neurodevelopmental disorders such as foetal alcohol spectrum disorder (FASD). This study aimed to examine macronutrient and micronutrient imbalances and assess the nutritional status of a group of patients with FASD. Methods: This study involved an analysis of the serum levels of key nutrients in a group of children diagnosed with FASD. Macronutrients and micronutrients were measured to identify any imbalances, including vitamin D, B12, E, A, albumin, and serum protein, among others. Results: The study found a high prevalence of vitamin D deficiency among the patients. Additionally, elevated serum concentrations of micronutrients such as vitamin B12, E, and A were observed in 8%, 7%, and 19% of patients, respectively. Macronutrient imbalances were noted, including high levels of albumin and serum protein, indicating a possible metabolic disturbance. Unexpectedly, high rates of hypercholesterolemia were observed, raising concerns about an increased risk of metabolic syndrome in this population. Conclusions: These findings suggest that the principal issue among patients with FASD is an altered metabolism rather than nutritional deficiencies. Potential causes of these abnormalities could include oxidative stress and changes in body composition. The results underline the need for further research to better understand the unique nutritional challenges in children with FASD and to guide the development of targeted therapeutic strategies.

Terracina S., Tarani L., Ceccanti M., Vitali M., Francati S., Lucarelli M., Venditti S., Verdone L., Ferraguti G., Fiore M.

Fetal alcohol spectrum disorders (FASD) represent a continuum of lifelong impairments resulting from prenatal exposure to alcohol, with significant global impact. The “spectrum” of disorders includes a continuum of physical, cognitive, behavioral, and developmental impairments which can have profound and lasting effects on individuals throughout their lives, impacting their health, social interactions, psychological well-being, and every aspect of their lives. This narrative paper explores the intricate relationship between oxidative stress and epigenetics in FASD pathogenesis and its therapeutic implications. Oxidative stress, induced by alcohol metabolism, disrupts cellular components, particularly in the vulnerable fetal brain, leading to aberrant development. Furthermore, oxidative stress is implicated in epigenetic changes, including alterations in DNA methylation, histone modifications, and microRNA expression, which influence gene regulation in FASD patients. Moreover, mitochondrial dysfunction and neuroinflammation contribute to epigenetic changes associated with FASD. Understanding these mechanisms holds promise for targeted therapeutic interventions. This includes antioxidant supplementation and lifestyle modifications to mitigate FASD-related impairments. While preclinical studies show promise, further clinical trials are needed to validate these interventions’ efficacy in improving clinical outcomes for individuals affected by FASD. This comprehensive understanding of the role of oxidative stress in epigenetics in FASD underscores the importance of multidisciplinary approaches for diagnosis, management, and prevention strategies. Continued research in this field is crucial for advancing our knowledge and developing effective interventions to address this significant public health concern.

Honda K., Hase H., Tanikawa S., Okawa K., Chen L., Yamaguchi T., Nakai M., Kitae K., Ago Y., Nakagawa S., Tsujikawa K.

Abstract Transfer RNA (tRNA) modification is essential for proper protein translation, as these modifications play important roles in several biological functions and disease pathophysiologies. AlkB homolog 8 (ALKBH8) is one of the nine mammalian ALKBH family molecules known to regulate selenoprotein translation through the modification of the wobble uridine (U34) in tRNA; however, its specific biological roles remain unclear. In this study, we investigated the role of ALKBH8 using Alkbh8-knockout (Albkh8−/−) mice, which were observed to have reduced 5-methoxycarbonylmethyluridine (mcm5U) and (S)-5-methoxycarbonylhydroxymethyluridine levels; notably, the mcm5U level was partially compensated only in the brain. The results of the novel object recognition test showed reduction in time to explore a novel object in Albkh8−/− mice; increased latency to fall in the rotarod performance test and latency to the immobility period in the forced swim test were also observed. These abnormal behaviors indicate dysfunction of the central nervous system. Furthermore, we observed reduced brain weight and ischemic pathological changes in the cerebral cortex and hippocampus in the form of weak eosin staining in the fiber tracts adjacent to the hippocampal cornu ammonis 1 region and an increase in pyramidal cells in the temporal lobe. Concordantly, we identified the differential expression of oxidative stress-related proteins and metabolites in the cerebral cortex and hippocampus using omics analyses. Finally, neurons and glial cells derived from Albkh8−/− mice show reduced mitochondrial membrane potential. Collectively, these findings indicate that ALKBH8 maintains neural function through an oxidative stress-regulatory mechanism.

Bhatia S., Bodenstein D., Cheng A.P., Wells P.G.

Oxoguanine glycosylase 1 (OGG1) is widely known to repair the reactive oxygen species (ROS)-initiated DNA lesion 8-oxoguanine (8-oxoG), and more recently was shown to act as an epigenetic modifier. We have previously shown that saline-exposed Ogg1 −/− knockout progeny exhibited learning and memory deficits, which were enhanced by in utero exposure to a single low dose of ethanol (EtOH) in both Ogg1 +/+ and −/− progeny, but more so in Ogg1 −/− progeny. Herein, OGG1-deficient progeny exposed in utero to a single low dose of EtOH or its saline vehicle exhibited OGG1- and/or EtOH-dependent alterations in global histone methylation and acetylation, DNA methylation and gene expression (Tet1 (Tet Methylcytosine Dioxygenase 1), Nlgn3 (Neuroligin 3), Hdac2 (Histone Deacetylase 2), Reln (Reelin) and Esr1 (Estrogen Receptor 1)) in fetal brains, and behavioural changes in open field activity, social interaction and ultrasonic vocalization, but not prepulse inhibition. OGG1- and EtOH-dependent changes in Esr1 and Esr2 mRNA and protein levels were sex-dependent, as was the association of Esr1 gene expression with gene activation mark histone H3 lysine 4 trimethylation (H3K4me3) and gene repression mark histone H3 lysine 27 trimethylation (H3K27me3) measured via ChIP-qPCR. The OGG1-dependent changes in global epigenetic marks and gene/protein expression in fetal brains, and postnatal behavioural changes, observed in both saline- and EtOH-exposed progeny, suggest the involvement of epigenetic mechanisms in developmental disorders mediated by 8-oxoG and/or OGG1. Epigenetic effects of OGG1 may be involved in ESR1-mediated gene regulation, which may be altered by physiological and EtOH-enhanced levels of ROS formation, possibly contributing to sex-dependent developmental disorders observed in Ogg1 knockout mice. The OGG1- and EtOH-dependent associations provide a basis for more comprehensive mechanistic studies to determine the causal involvement of oxidative DNA damage and epigenetic changes in ROS-mediated neurodevelopmental disorders.

Lafuente J.V., Sharma A., Feng L., Muresanu D.F., Nozari A., Tian Z.R., Buzoianu A.D., Sjöquist P., Wiklund L., Sharma H.S.

Military personnel are often exposed to hot environments either for combat operations or peacekeeping missions. Hot environment is a severe stressful situation leading to profound hyperthermia, fatigue and neurological impairments. To avoid stressful environment, some people frequently use methamphetamine (METH) or other psychostimulants to feel comfortable under adverse situations. Our studies show that heat stress alone induces breakdown of the blood–brain barrier (BBB) and edema formation associated with reduced cerebral blood flow (CBF). On the other hand, METH alone induces hyperthermia and neurotoxicity. These effects of METH are exacerbated at high ambient temperatures as seen with greater breakdown of the BBB and brain pathology. Thus, a combination of METH use at hot environment may further enhance the brain damage-associated behavioral dysfunctions. METH is well known to induce severe oxidative stress leading to brain pathology. In this investigation, METH intoxication at hot environment was examined on brain pathology and to explore suitable strategies to induce neuroprotection. Accordingly, TiO2-nanowired delivery of H-290/51 (150 mg/kg, i.p.), a potent chain-breaking antioxidant in combination with mesenchymal stem cells (MSCs), is investigated in attenuating METH-induced brain damage at hot environment in model experiments. Our results show that nanodelivery of H-290/51 with MSCs significantly enhanced CBF and reduced BBB breakdown, edema formation and brain pathology following METH exposure at hot environment. These observations are the first to point out that METH exacerbated brain pathology at hot environment probably due to enhanced oxidative stress, and MSCs attenuate these adverse effects, not reported earlier.

Ibrahim A., Warton F.L., Fry S., Cotton M.F., Jacobson S.W., Jacobson J.L., Molteno C.D., Little F., van der Kouwe A.J., Laughton B., Meintjes E.M., Holmes M.J.

IntroductionSuccessful programmes for prevention of vertical HIV transmission have reduced the risk of infant HIV infection in South Africa from 8% in 2008 to below 1% in 2018/2019, resulting in an increasing population of children exposed to HIV perinatally but who are uninfected (HEU). However, the long-term effects of HIV and antiretroviral treatment (ART) exposure on the developing brain are not well understood. Whereas children who are HEU perform better than their HIV-infected counterparts, they demonstrate greater neurodevelopmental delay than children who are HIV unexposed and uninfected (HUU), especially in resource-poor settings. Here we investigate subcortical volumetric differences related to HIV and ART exposure in neonates.MethodsWe included 120 infants (59 girls; 79 HEU) born to healthy women with and without HIV infection in Cape Town, South Africa, where HIV sero-prevalence approaches 30%. Of the 79 HEU infants, 40 were exposed to ART throughout gestation (i.e., mothers initiated ART pre conception; HEU-pre), and 39 were exposed to ART for part of gestation (i.e., mothers initiated ART post conception; HEU-post). Post-conception mothers had a mean (± SD) gestational age (GA) of 15.4 (± 5.7) weeks at ART initiation. Mothers with HIV received standard care fixed drug combination ART (Tenofovir/Efavirenz/Emtricitabine). Infants were imaged unsedated on a 3T Skyra (Siemens, Erlangen, Germany) at mean GA equivalent of 41.5 (± 1.0) weeks. Selected regions (caudate, putamen, pallidum, thalamus, cerebellar hemispheres and vermis, and corpus callosum) were manually traced on T1-weighted images using Freeview.ResultsHEU neonates had smaller left putamen volumes than HUU [β (SE) = −90.3 (45.3), p = 0.05] and caudate volume reductions that depended on ART exposure duration in utero. While the HEU-pre group demonstrated no caudate volume reductions compared to HUU, the HEU-post group had smaller caudate volumes bilaterally [β (SE) = −145.5 (45.1), p = 0.002, and −135.7 (49.7), p = 0.008 for left and right caudate, respectively].DiscussionThese findings from the first postnatal month suggest that maternal ART throughout gestation is protective to the caudate nuclei. In contrast, left putamens were smaller across all HEU newborns, despite maternal ART.

D’aloisio G., Acevedo M.B., Angulo-Alcalde A., Trujillo V., Molina J.C.

Early ontogeny of the rat (late gestation and postnatal first week) is a sensitive period to ethanol’s positive reinforcing effects and its detrimental effects on respiratory plasticity. Recent studies show that acetaldehyde, the first ethanol metabolite, plays a key role in the modulation of ethanol motivational effects. Ethanol brain metabolization into acetaldehyde via the catalase system appears critical in modulating ethanol positive reinforcing consequences. Catalase system activity peak levels occur early in the ontogeny. Yet, the role of ethanol-derived acetaldehyde during the late gestational period on respiration response, ultrasonic vocalizations (USVs), and ethanol intake during the first week of the rat remains poorly explored. In the present study, pregnant rats were given a subcutaneous injection of an acetaldehyde-sequestering agent (D-penicillamine, 50 mg/kg) or saline (0.9% NaCl), 30 min prior to an intragastric administration of ethanol (2.0 g/kg) or water (vehicle) on gestational days 17–20. Respiration rates (breaths/min) and apneic episodes in a whole-body plethysmograph were registered on postnatal days (PDs) 2 and 4, while simultaneously pups received milk or ethanol infusions for 40-min in an artificial lactation test. Each intake test was followed by a 5-min long USVs emission record. On PD 8, immediately after pups completed a 15-min ethanol intake test, brain samples were collected and kept frozen for catalase activity determination. Results indicated that a moderate experience with ethanol during the late gestational period disrupted breathing plasticity, increased ethanol intake, as well brain catalase activity. Animals postnatally exposed to ethanol increased their ethanol intake and exerted differential affective reactions on USVs and apneic episodes depending on whether the experience with ethanol occur prenatal or postnatally. Under the present experimental conditions, we failed to observe, a clear role of acetaldehyde mediating ethanol’s effects on respiratory plasticity or affective states, nevertheless gestational acetaldehyde was of crucial importance in determining subsequent ethanol intake affinity. As a whole, results emphasize the importance of considering the participation of acetaldehyde in fetal programming processes derived from a brief moderate ethanol experience early in development, which in turn, argues against “safe or harmless” ethanol levels of exposure.

Fainsod A., Abbou T., Bendelac-Kapon L., Edri T., Pillemer G.

Multiple models were proposed to explain the mechanism(s) of alcohol (ethanol) teratogenesis inducing the wide range of developmental defects, neurobehavioral anomalies, and mental disabilities known collectively as Fetal Alcohol Spectrum Disorder (FASD). Competition between alcohol clearance and retinoic acid (RA) biosynthesis was proposed as both processes employ the same families of enzymes. Excess of ethanol or its clearance metabolite, acetaldehyde, will compete with vitamin A (retinol) or retinaldehyde and hamper the production of RA with teratogenic outcomes. Taking advantage of the ease of manipulation, external development, and large clutch sizes in Xenopus , we have been studying and characterizing the alcohol/RARetinoic acid (RA) competition modelCompetition model. Xenopus embryosEmbryos recapitulate many of the developmentalDevelopmental malformations of Fetal Alcohol Syndrome (FASFetal alcohol syndrome (FAS)), the more severe form of FASDFetal alcohol spectrum disorder (FASD). The effect of ethanol on development is most severe during gastrulaGastrula stages and continues, but with milder outcomes throughout development. Ethanol targets the “embryonic organizerEmbryonic organizer,” the earliest site of RARetinoic acid (RA) signaling. To support the connection between ethanol and RARetinoic acid (RA), we show that all abnormal embryonic processes or molecular events induced by ethanol can be reproduced by reducing RARetinoic acid (RA) signaling levels. Importantly, the effects of ethanol can be rescued by increasing RARetinoic acid (RA) signaling, and RARetinoic acid (RA) reduction hypersensitizes the embryoEmbryos to alcohol exposure. Biochemical studies demonstrated that RARetinoic acid (RA) biosynthetic enzymes can readily function in ethanol clearance. Additional syndromes linked to reduced RARetinoic acid (RA) signaling with partially overlapping phenotypes with FASDFetal alcohol spectrum disorder (FASD) are discussed.

Carey M.E., Rando J., Melnyk S., James S.J., Snyder N., Salafia C., Croen L.A., Fallin M.D., Hertz-Picciotto I., Volk H., Newschaffer C., Lyall K.

We examined associations between prenatal oxidative stress (OS) and child autism-related outcomes. Women with an autistic child were followed through a subsequent pregnancy and that younger sibling’s childhood. Associations between glutathione (GSH), glutathione disulfide (GSSG), 8-oxo-deoxyguanine (8-OHdG), and nitrotyrosine and younger sibling Social Responsiveness Scale (SRS) scores were examined using quantile regression. Increasing GSH:GSSG (suggesting decreasing OS) was associated with minor increases in SRS scores (50th percentile β: 1.78, 95% CI: 0.67, 3.06); no other associations were observed. Results from this cohort with increased risk for autism do not support a strong relationship between OS in late pregnancy and autism-related outcomes. Results may be specific to those with enriched autism risk; future work should consider other timepoints and biomarkers.

Pinto-Ribeiro L., Silva C., Andrade N., Martel F.

Extravillous trophoblasts (EVTs) are the main participants in the process of placentation, an early process critical for placental growth and function involving an adequate invasion and complete remodelling of the maternal spiral arteries during early pregnancy. An increase in oxidative stress during pregnancy is associated with the onset and progression of several pregnancy disorders, including preeclampsia and gestational diabetes mellitus and it also occurs due to exposure of pregnant women to some xenobiotics (eg. alcohol). This study aimed to investigate how oxidative stress affects EVTs, and the ability of several distinct antioxidant agents to prevent these changes. For this, we exposed HTR8/SVneo cells to tert-butylhydroperoxide (0.5 μM; 24 h), which was able to increase lipid peroxidation and protein carbonyl levels. Under these conditions, there was a decrease in proliferation rates, culture growth, migratory and angiogenic capacities and an increase in the apoptosis rates. The antiproliferative effect of TBH was supressed by simultaneous treatment of the cells with α-tocopherol, but other antioxidants (vitamin C, allopurinol, apocynin, N-acetylcysteine, quercetin and resveratrol) were ineffective. α-tocopherol was also able to abolish the effect of TBH on lipid peroxidation and protein carbonyl levels. Overall, our results show that oxidative stress interferes with EVT characteristics essential for the placentation process, which may contribute to the association between oxidative stress and pregnancy disorders. Our results also show that the nature of the in vitro model of oxidative stress-induction is an important determinant of the cellular consequences of oxidative stress and, therefore, of the efficacy of antioxidants.

Bhatia S., Arslan E., Rodriguez-Hernandez L.D., Bonin R., Wells P.G.

Abstract Oxoguanine glycosylase 1 (OGG1) repairs the predominant reactive oxygen species-initiated DNA lesion 8-oxoguanine. Human OGG1 polymorphisms resulting in reduced DNA repair associate with an increased risk for disorders like cancer and diabetes, but the role of OGG1 in brain development is unclear. Herein, we show that Ogg1 knockout mice at 2–3 months of age exhibit enhanced gene- and sex-dependent DNA damage (strand breaks) and decreased epigenetic DNA methylation marks (5-methylcytosine, 5-hydroxymethylcytosine), both of which were associated with increased cerebellar calbindin levels, reduced hippocampal postsynaptic function, altered body weight with age and disorders of brain function reflected in behavioral tests for goal-directed repetitive behavior, anxiety and fear, object recognition and spatial memory, motor coordination and startle response. These results suggest that OGG1 plays an important role in normal brain development, possibly via both its DNA repair activity and its role as an epigenetic modifier, with OGG1 deficiencies potentially contributing to neurodevelopmental disorders.

Ferraguti G., Terracina S., Petrella C., Greco A., Minni A., Lucarelli M., Agostinelli E., Ralli M., de Vincentiis M., Raponi G., Polimeni A., Ceccanti M., Caronti B., Di Certo M.G., Barbato C., et. al.

Head and neck cancer (HNC) concerns more than 890,000 patients worldwide annually and is associated with the advanced stage at presentation and heavy outcomes. Alcohol drinking, together with tobacco smoking, and human papillomavirus infection are the main recognized risk factors. The tumorigenesis of HNC represents an intricate sequential process that implicates a gradual acquisition of genetic and epigenetics alterations targeting crucial pathways regulating cell growth, motility, and stromal interactions. Tumor microenvironment and growth factors also play a major role in HNC. Alcohol toxicity is caused both directly by ethanol and indirectly by its metabolic products, with the involvement of the oral microbiota and oxidative stress; alcohol might enhance the exposure of epithelial cells to carcinogens, causing epigenetic modifications, DNA damage, and inaccurate DNA repair with the formation of DNA adducts. Long-term markers of alcohol consumption, especially those detected in the hair, may provide crucial information on the real alcohol drinking of HNC patients. Strategies for prevention could include food supplements as polyphenols, and alkylating drugs as therapy that play a key role in HNC management. Indeed, polyphenols throughout their antioxidant and anti-inflammatory actions may counteract or limit the toxic effect of alcohol whereas alkylating agents inhibiting cancer cells’ growth could reduce the carcinogenic damage induced by alcohol. Despite the established association between alcohol and HNC, a concerning pattern of alcohol consumption in survivors of HNC has been shown. It is of primary importance to increase the awareness of cancer risks associated with alcohol consumption, both in oncologic patients and the general population, to provide advice for reducing HNC prevalence and complications.

Uetrecht J., Grant D.M., Wells P.G.

Drugs are an essential tool for the treatment of many diseases; however, drugs can also cause many different types of adverse effects. Many adverse reactions are simply an extension of their therapeutic effects and are relatively easy to predict. The focus of this chapter will be on idiosyncratic adverse drug reactions, carcinogenic effects of drugs, and teratogenic effects of drugs. Drugs can be converted to multiple metabolites, and these metabolites can be responsible for many of the adverse effects associated with the use of medication. In particular, chemically reactive metabolites can cause both direct toxicity such as carcinogenic and teratogenic effects, and they can also result in adverse immune responses. A better mechanistic understanding of adverse drug reactions would lead to safer use of current drugs and make it easier to screen drug candidates for their potential to cause adverse reactions.

Fiore M., Petrella C., Coriale G., Rosso P., Fico E., Ralli M., Greco A., De Vincentiis M., Minni A., Polimeni A., Vitali M., Messina M.P., Ferraguti G., Tarani F., de Persis S., et. al.

Background: Fetal alcohol spectrum disorders (FASD) are the manifestation of the damage caused by alcohol consumption during pregnancy. Children with fetal alcohol syndrome (FAS), the extreme FASD manifestation, show both facial dysmorphology and mental retardation. Alcohol consumed during gestational age prejudices brain development by reducing, among others, the synthesis and release of neurotrophic factors and neuroinflammatory markers. Alcohol drinking also induces oxidative stress. Hypothesis/Objective: The present study aimed to investigate the potential association between neurotrophins, neuroinflammation, and oxidative stress in 12 prepubertal male and female FASD children diagnosed as FAS or partial FAS (pFAS). Methods: Accordingly, we analyzed, in the serum, the level of BDNF and NGF and the oxidative stress, as free oxygen radicals test (FORT) and free oxygen radicals defense (FORD). Moreover, serum levels of inflammatory mediators (IL-1α, IL-2, IL-6, IL-10, IL-12, MCP-1, TGF-β, and TNF- α) involved in neuroinflammatory and oxidative processes have been investigated. Results: We demonstrated low serum levels of NGF and BDNF in pre-pubertal FASD children with respect to healthy controls. These changes were associated with higher serum presence of TNF- α and IL-1α. Quite interestingly, an elevation in the FORD was also found despite normal FORT levels. Moreover, we found a potentiation of IL-1α, IL-2, IL-10, and IL-1α1 in the analyzed female compared to male children. Conclusion: The present investigation shows an imbalance in the peripheral neuroimmune pathways that could be used in children as early biomarkers of the deficits observed in FASD.